Page 1 — Project overview, system size, and materials 🧫🏭

Main idea

The project presents an imaginary design for producing insulin using the yeast Pichia pastoris in a fed-batch fermentation system.

The goal is not to build a fully validated real plant, but to outline:

• Main equipment
• Process steps
• Material choices
• Economic estimates
• A rough design for a 20 m³ fermentation system
• Downstream purification assumptions

The authors are very clear that the project contains many assumptions because they lack real process-specific data such as:

• Growth rate
• Agitation rate
• Oxygen transfer efficiency
• Actual insulin productivity
• Purification yield
• Required purity
• Industrial validation requirements

A key honest point is that if this were a real startup project, the correct strategy would be to begin at lab scale, collect reliable experimental data, and only then gradually scale up.

System dimensions

The process is designed around a large production vessel supported by smaller seed vessels.

The fermentation scale-up train includes:

• 1 L shake flasks
• 10 L seed fermenter
• 100 L seed fermenter
• 1000 L seed fermenter
• 20 m³ main fermenter

The largest vessel is the main production bioreactor, while the smaller vessels are used to grow enough cells to inoculate the main vessel.

Supporting tanks

The project also includes several tanks:

• Feed tank: 30 m³
• Acid tank: 3.0 m³
• Base tank: 3.0 m³
• Methanol inducer tank: 0.1 m³
• Anti-foam tank: 0.1 m³
• Hot water tank: 1 m³
• Cold water tank: 1 m³

Methanol is included because Pichia pastoris expression systems often use methanol-inducible promoters, especially the AOX1 promoter system.

Gas supply

Most oxygen is supplied using atmospheric air, with additional oxygen from an oxygen tank when needed.

This makes sense because high-density yeast fermentation can become oxygen-limited. Air is cheaper, but pure oxygen supplementation may be necessary when oxygen demand becomes too high.

Piping and equipment count

The design estimates around:

• 600 meters of stainless steel pipe
• 11 pumps
• 16 valves
• 8 controllers/indicators/transmitters
• 2 water heaters
• 2 compressors
• 1 fan
• 1 air filter

These are included in the P&ID.

Material choices

Most equipment must be made from 316L stainless steel, except the external water system.

That is important because 316L stainless steel is commonly used in pharmaceutical and food-grade systems due to:

• Corrosion resistance
• Cleanability
• Compatibility with CIP/SIP systems
• Lower carbon content compared with regular 316 stainless steel
• Suitability for sanitary design

The acid/base parts need extra protection:

• Acid/base pumps, pipes, and valves should be PTFE-lined
• This protects them from chemical corrosion

Page 2 — P&ID diagram, pumps, and process layout ⚙️📈

P&ID overview

Page 2 contains a simplified piping and instrumentation diagram for the 20 m³ fermenter and supporting equipment.

The diagram shows the main fermenter connected to:

• Inoculum line
• Feed tank
• Methanol inducer tank
• Acid tank
• Base tank
• Anti-foam tank
• Oxygen tank
• Air intake and sterile filter
• Condenser/off-gas line
• Hot/cold jacket water system
• Downstream processing outlet

The diagram also shows multiple control loops, including:

• Temperature control
• pH control
• Dissolved oxygen control
• Optical density control
• Level control
• Pressure control

In simple terms, the diagram describes a controlled bioreactor system where nutrients, pH correction chemicals, oxygen, temperature regulation, and foam control are automatically adjusted.

External/jacket water system pumps 💧

The external water system is used for temperature control through the fermenter jacket.

The listed pumps are:

External water source pump

A centrifugal pump used for bulk transport of water.

It needs a relatively high flow rate because it supplies water to the water system.

Cold water pump

Also centrifugal, used to move cold water through the system.

Jacket water outlet pump

Used to remove water from the jacket loop.

Hot water pump

A centrifugal pump with heat resistance, because it handles hot water.

Fermenter vessel pumps 🧪

These pumps interact more directly with the sterile fermentation process, so they need more careful design.

Inoculum pump

A sanitary peristaltic pump.

It should be gentle because the inoculum contains living cells, and excessive shear stress could damage them.

Feed pump

A sanitary diaphragm pump.

It must deliver a steady and precise flow, because fed-batch fermentation depends heavily on controlled nutrient addition.

Inducer pump

A sanitary peristaltic pump.

It requires low-flow precision, because methanol addition must be carefully controlled. Too much methanol can become toxic or create safety risks.

Anti-foam pump

Also a sanitary peristaltic pump.

It only needs to add small amounts of anti-foam when foam becomes problematic.

Page 3 — More pumps and full valve list 🚰🔩

Page 3 continues the equipment list, especially pumps and valves.

Additional fermenter pumps

Acid pump

A sanitary diaphragm pump.

It must be chemically resistant and respond quickly when the pH becomes too high.

Base pump

Also a sanitary diaphragm pump.

It must be chemically resistant and respond quickly when the pH becomes too low.

Downstream processing pump

This pump transfers the fermentation broth to downstream purification at the end of the batch.

It must be gentle enough to avoid unnecessary cell/product damage, but much more powerful than the inoculum pump because it must empty the full vessel.

Valve list

The system includes many valve types, each chosen based on sterility, pressure, chemical resistance, or control requirements.

Atmospheric air valve

A sanitary diaphragm valve.

This is connected to sterile air filtration, so sterility is critical.

O₂ tank valve

A high-pressure stainless steel diaphragm valve.

The document correctly notes that lubricated mechanical parts are dangerous around pure oxygen because they can create a fire hazard.

Vessel pressure safety valve

A spring-loaded hygienic pressure relief valve.

Its purpose is safety: preventing overpressure in the fermenter.

Vessel air outflow valve

A sanitary diaphragm valve.

Because off-gas may contain microbes, the system must prevent contamination of the environment.

Antifoam valve

A level-controlled sanitary diaphragm valve.

It opens when foam reaches a safety level, assuming the level increase is due to foaming.

Downstream processing valve

A level-controlled sanitary diaphragm valve.

It opens automatically when the vessel reaches a certain level threshold.

Jacket outlet valve

A three-way ball valve.

It directs jacket water either to wastewater or back to the hot water tank depending on temperature.

External water source valve

A simple ball valve.

Sterility is not a major concern here because this is part of the external water system.

Hot/cold combination valve

A three-way globe valve.

This helps precisely mix hot and cold water for temperature control.

Hot water backflow / steam water inflow valve

A globe control valve.

It must tolerate high temperature and help with precise temperature regulation.

Steam tank pressure safety valve

Protects the steam tank from dangerous pressure buildup.

Steam inflow valve into vessel

A manual globe valve that must tolerate steam and avoid leakage.

Feed valve

A sanitary diaphragm valve controlled by optical density.

It opens when OD reaches a certain threshold, meaning feed is added based on cell growth.

Inducer valve

Also OD-controlled.

This is used for methanol induction, likely when biomass is high enough for recombinant insulin production.

Page 4 — Acid/base valves and pump CAPEX 💰⚙️

Acid and base valves

The acid and base valves are both PTFE-lined sanitary diaphragm valves.

They are controlled by pH:

• Acid valve opens when pH is too high
• Base valve opens when pH is too low

They must be:

• Sanitary
• Chemically resistant
• Accurate
• Automated

This is important because pH strongly affects yeast growth, protein expression, enzyme activity, and product stability.

Pump costs

Page 4 begins the CAPEX section: capital expenditure, meaning the cost of buying/building the system.

Water pumps

The water pumps are estimated at 23,400 kr total.

The external pump has:

• Flow rate: 11 m³/h
• Head: 8 m

The other water pumps have:

• Flow rate: 9 m³/h
• Head: 6 m

Sterile vessel pumps

The document estimates several sanitary pump prices:

Inoculum pumps

• 10 pumps
• 21,300 kr each
• Max flow rate: 144 L/h

The group uses 10 pumps because one pump alone would take about 420 minutes to transfer inoculum, while 10 pumps reduce this to about 42 minutes.

A skeptical note: putting pumps “in series” does not generally multiply flow rate the same way putting pumps in parallel would. Pumps in series usually increase pressure/head, while pumps in parallel increase total flow. So the design idea is directionally understandable, but the engineering wording may be flawed.

Antifoam / inducer pumps

• 37,500 kr each
• Max flow rate: 0.03 m³/h
• Max head: 71 m

These are low-flow precision pumps.

Feed pump

• 26,900 kr
• Max flow rate: 5.45 m³/h
• Max head: 30 m

Acid/base pumps

• 41,300 kr each
• Max flow rate: 24 m³/h
• Max pressure: about 7 bar

Downstream pump

• 57,400 kr
• Max flow rate: 115 m³/h
• Pressure: 15 bar

Pump price uncertainty

The authors note that many industry-standard sanitary pump prices are not public and require formal supplier quotes.

They also admit that some selected pumps may not be truly pharmaceutical grade.

To compensate, they add a 40% overhead.

Final pump estimate:

• Base estimate: 478,300 kr
• With 40% overhead: ~670,000 kr

Page 5 — Valves, piping, vessels, and tanks 🧱🏭

Valve CAPEX

The group could not find reliable pharmaceutical-grade valve prices, so they used cheaper online listings as a starting point.

They found valve prices between:

• 500 kr
• 2,600 kr

They chose the higher estimate:

• 2,600 kr per valve

Then they multiplied by 6 to approximate pharmaceutical-grade pricing.

Calculation:

• 16 valves × 2,600 kr × 6 = 249,600 kr

Pipes

The design assumes:

• 600 meters
• 2-inch 316L stainless steel pipe

The raw material cost is estimated at:

• 428,000 kr

Because the pipe surface roughness is not specified, they add 40% for:

• Electropolishing
• Certification
• Achieving approximately 0.8 Ra surface roughness

That matters because smoother surfaces are easier to clean and reduce microbial/product buildup.

Final pipe estimate:

• 1.4 × 428,000 kr = 599,200 kr

Vessels

The vessel system includes:

Total before overhead:

• 7,146,000 kr

With 40% overhead:

• ~10,000,000 kr

One small inconsistency: earlier the document describes the largest fermentation vessel as 20 m³, but the CAPEX section lists a 30 m³ main reactor. This may reflect required working volume versus total vessel volume, but the document does not fully clarify it.

Tanks

The tank costs include:

Total before overhead:

• 279,600 kr

With 40% overhead:

• ~391,500 kr

Page 6 — CIP/SIP, control systems, Lang factor, and fermentation CAPEX 🧼🧠

CIP/SIP system

CIP means clean-in-place.

SIP means sterilize-in-place.

These systems are critical in pharmaceutical production because equipment must be cleaned and sterilized without disassembly.

The authors correctly state that designing a full CIP/SIP system could be an entire engineering project by itself.

They use a supplier listing for a pharmaceutical aseptic CIP/SIP unit:

• Base estimate: 1,300,000 kr

Because they doubt it would meet European insulin-production standards, they add 40%.

Final CIP/SIP estimate:

• 1,820,000 kr

Control systems

The plant needs automated control systems that coordinate signals from:

• pH sensors
• OD sensors
• Level sensors
• Temperature sensors
• Pressure sensors
• Dissolved oxygen sensors
• Pumps
• Valves

The document mentions a PLC estimate of:

• 767,000 kr

But it also notes that a real plant would need much more than just a PLC, including:

• Redundant DCS
• SCADA
• Batch orchestration software
• Instrumentation and I/O expansion
• Control cabinets
• Validated wiring
• GMP documentation
• Installation and validation

This is a strong point in the report: for pharmaceutical production, automation cost is not only hardware. Validation and compliance can be extremely expensive.

Lang factor estimate

The group uses a Lang factor to estimate total capital investment.

Instead of pricing every installation detail individually, they take the main equipment costs and multiply by a factor.

They use:

• Lang factor = 6

The usual factor for a fluid processing factory is listed as 4.74, but they use 6 to account for pharmaceutical-grade requirements.

Main fermentation components:

• Pumps: 670,000 kr
• Valves: 250,000 kr
• Pipes: 600,000 kr
• Vessels: 10,000,000 kr
• Tanks: 392,000 kr
• CIP/SIP: 1,820,000 kr
• PLC: 767,000 kr

Final fermentation capital investment estimate:

• ~87,000,000 DKK

Start of downstream section

The page ends by introducing downstream purification systems.

The authors note that estimating downstream purification cost is difficult because pricing depends heavily on:

• Vendor
• Automation level
• Scale
• Regulatory requirements
• Resin choice
• Column dimensions
• Validation demands

Page 7 — Downstream purification CAPEX 🧪💎

Page 7 estimates the capital investment for purification equipment.

This is the downstream part of insulin production: after fermentation, the product must be separated, purified, processed, polished, crystallized, and formulated.

The document estimates a large uncertainty of ±30–50%, which is realistic for this kind of early-stage estimate.

Purification equipment list

Total purification capital investment:

• 79,000,000 DKK

What this purification train does

The process appears to assume production of a crude insulin or proinsulin-like product from Pichia pastoris, followed by several purification and conversion steps.

The likely logic is:

1. Remove cells
2. Concentrate product
3. Capture insulin/proinsulin
4. Remove major impurities
5. Convert precursor into mature insulin
6. Polish away remaining impurities
7. Crystallize insulin for stable final form

Page 8 — Total CAPEX and beginning of OPEX 📊💸

Total CAPEX

The total capital expenditure combines fermentation and purification:

• Fermentation: 87,000,000 DKK
• Purification: 79,000,000 DKK

Total CAPEX:

• 166,000,000 DKK

This is the estimated equipment/buildout investment before operating costs and loan assumptions.

OPEX overview

OPEX means operating expenses: the cost to run each batch.

The document estimates costs per batch.

Feed cost

Media/feed cost is estimated as:

• 208,929.6 DKK per batch

This assumes:

• 24 m³ media
• 8.7054 DKK/L

Electricity cost

Total electricity cost is listed as:

• 23,020 DKK per batch

This includes cooling and agitation for a batch producing 8 kg pure insulin.

Pump electricity is estimated separately:

• 1 pump consumes 1.5 kW
• 15 pumps total
• Pumps operate 20% of the time
• Total pump electricity: 225 kWh/batch
• Electricity price: 2.5 DKK/kWh
• Pump electricity cost: 562.5 DKK/batch

So total electricity including pump electricity becomes:

• 23,582.5 DKK/batch

Chemical costs

The system assumes use of acid and base to control pH between 6.3 and 6.5.

The acid/base system uses:

• H₃PO₄, 25% v/v
• NH₄OH, 25% v/v

The document assumes 4000 L total acid/base consumption, split equally between acid and base.

Costs:

• Acid: 21,040.42 DKK/batch
• Base: 6,370.04 DKK/batch

Combined pH adjustment cost:

• 27,410.0 DKK/batch

A critical note: 4000 L of acid/base for a 20 m³ fermenter is a very large assumption. It may be possible in some processes, but it would need experimental justification.

Labor cost

Labor is estimated as 20% of revenue.

The document gives:

• Labor cost: 261,251.58 DKK/batch

But later, the OPEX summary uses 157,251.58 DKK/batch instead. That is an internal inconsistency.

Waste disposal

Waste per batch:

• 60 m³

Waste treatment cost:

• 55 DKK/m³

Total waste disposal:

• 3,300 DKK/batch

Start of purification OPEX

Page 8 also begins explaining purification operating costs.

The first step is:

Cell harvest and clarification

Equipment/process:

• Disc-stack centrifugation
• Depth filtration

Estimated batch cost:

• 120,000–150,000 DKK

Expected yield:

• 98–99%

Purpose:

• Remove yeast biomass from the culture medium
• Use centrifugation to separate cells
• Use depth filtration to remove remaining particles

Page 9 — Purification operating steps and yields 🔬🧴

Page 9 continues the downstream purification OPEX and explains what each step does.

UF/DF concentration

UF/DF means ultrafiltration/diafiltration.

Estimated cost:

• 150,000–225,000 DKK

Expected yield:

• 95–96%

Purpose:

• Concentrate insulin/proinsulin
• Exchange buffer
• Remove some contaminants
• Prepare the product for chromatography

The document mentions 100 kDa + 10 kDa PES cassettes.

Cation exchange chromatography

CEX means cation exchange chromatography.

Estimated cost:

• 375,000–525,000 DKK

Expected yield:

• 85–90%

Purpose:

• Capture/select target protein
• Reduce process volume
• Separate insulin from charged impurities
• Remove smaller peptides and aggregates

CEX separates molecules based on charge. Positively charged proteins bind to negatively charged resin.

Intermediate purification: HIC and mixed-mode chromatography

Estimated cost:

• 375,000–600,000 DKK

Expected yield:

• 90%

HIC means hydrophobic interaction chromatography.

Purpose:

• Remove product-related impurities
• Remove high-molecular-weight aggregates
• Separate molecules based on surface hydrophobicity

Mixed-mode chromatography combines multiple separation principles, such as charge and hydrophobicity.

Enzymatic processing and cleavage

Estimated cost:

• 150,000–300,000 DKK

Expected yield:

• 90–95%

Purpose:

• Convert inactive single-chain precursor/proinsulin into mature active insulin

Enzymes used:

• Trypsin
• Carboxypeptidase B

This mimics natural insulin maturation, where proinsulin is processed into insulin by removing connecting peptide regions.

AEX polishing and sterile filtration

AEX means anion exchange chromatography.

Estimated cost:

• 75,000–150,000 DKK

Expected yield:

• 95%

Purpose:

• Final impurity removal
• Remove negatively charged contaminants
• Remove host cell proteins
• Remove DNA/RNA
• Remove insulin-related variants, such as desamido-insulin
• Prepare product for sterile filtration

Sterile filtration is typically one of the final steps before aseptic filling.

Page 10 — Crystallization, total OPEX, profit, and rent 📦💰

Crystallization

Estimated cost:

• 40,000–47,000 DKK

Expected yield:

• 90%

Purpose:

• Convert liquid insulin into a stable high-purity solid form
• Usually zinc-insulin crystals
• Improve storage stability
• Prepare for final formulation

Purification summary

The total estimated purification operating cost is:

• 1,280,000–2,000,000 DKK

The document uses:

• 1,500,000 DKK per batch

Expected total purification yield:

• 40–50%

The assumed production result is:

• 20 kg crude product
• 40% recovery
• 8 kg pure insulin

OPEX per batch

The batch duration is estimated as:

• 50 hours

Final batch yield:

• 8 kg pure insulin

OPEX breakdown:

Total OPEX without labor:

• 1,757,742.1 DKK/batch

OPEX with labor:

• 1,914,993.7 DKK/batch

But as mentioned earlier, the labor number has an inconsistency: the page earlier says 261,251.58 DKK/batch, while the OPEX equation uses 157,251.58 DKK/batch.

Revenue

Selling price of insulin:

• 318 DKK/g

Pure insulin produced:

• 8 kg = 8000 g

Revenue:

• 8000 g × 318 DKK/g = 2,544,000 DKK/batch

Rent cost

The plant is assumed to require:

• 15,000 m²

Rent assumption:

• 1,200 DKK/m²/year

Annual rent:

• 18,000,000 DKK/year

Monthly rent:

• 1,500,000 DKK/month

Profit per batch

Net profit before rent:

• 2,544,000 − 1,914,993.7 = 629,006.3 DKK/batch

Monthly production assumption:

• 8 batches/month

Monthly profit before rent:

• 629,006.3 × 8 = 5,032,050.4 DKK/month

After rent:

• 5,032,050.4 − 1,500,000 = 3,532,050.4 DKK/month

Page 11 — Loan, repayment scenarios, tax, and ROI 🏦📉

Total investment request

The document estimates the required loan/requested investment as:

• CAPEX: 166,000,000 DKK
• Plus two months of operating costs with multiplier:
  • 1,914,993.7 × 2 = 3,829,987.4 DKK

Total requested loan:

• 169,829,987.4 DKK

Scenario 1 — Long-term loan

Assumptions:

• Interest rate: 5%
• Loan repayment period: 30 years
• Monthly repayment: 900,043.80 DKK

Profit before loan payment and tax:

• 3,532,050.4 DKK/month

Profit after loan payment:

• 3,532,050.4 − 900,043.80 = 2,632,006.6 DKK/month

Corporate tax assumption:

• 22%

Profit after tax:

• 2,632,006.6 × 0.78 = 2,052,965.148 DKK/month

So in Scenario 1, the company still makes about:

• 2.05 million DKK/month after loan payment and tax

Scenario 2 — Fast repayment

Assumptions:

• Interest rate: 5%
• Full repayment over 4 years and 6 months
• Monthly payment: 2,632,006.6 DKK
• Profit during repayment: 0 DKK/month

After the loan is fully repaid, the monthly profit after tax becomes:

• 3,532,050.4 × 0.78 = 2,754,999.312 DKK/month

So Scenario 2 sacrifices short-term profit to eliminate the loan much faster.

Overall summary — What the whole project is saying 🧠✨

This project designs a hypothetical industrial insulin production system using Pichia pastoris. It includes a 20 m³ fermentation process, seed train, feed/acid/base/methanol/anti-foam tanks, oxygen supply, water-jacket temperature control, sterile pumping, valves, CIP/SIP, automation, and downstream purification.

The estimated costs are:

The project’s biggest strengths are:

• It gives a full process overview from fermentation to purification.
• It includes P&ID thinking, not just biology.
• It considers pumps, valves, tanks, pipes, materials, and automation.
• It includes both CAPEX and OPEX.
• It honestly acknowledges uncertainty.
• It includes purification yield and downstream costs, which are often major cost drivers.

The biggest weak points or assumptions are:

• Many equipment prices are rough estimates from non-pharmaceutical sources.
• The insulin productivity assumption of 1 g/L crude product needs experimental support.
• The 40% purification yield is plausible as a rough number, but needs justification.
• The labor cost is inconsistent between pages.
• The main reactor is described as both 20 m³ and 30 m³, which should be clarified.
• Pump scaling using “10 pumps in series” may be technically incorrect if the goal is higher flow.
• Regulatory costs, GMP validation, QA/QC, facility qualification, cleaning validation, and batch release testing are probably underestimated.
• Rent and facility size assumptions may be oversimplified.
• Selling all product at 318 DKK/g assumes full market access and regulatory approval, which is a major real-world barrier.

Key takeaway 🧬🏭

The document is a useful early conceptual design for a Pichia pastoris-based insulin plant. It gives a broad picture of what equipment and economics might look like, but it should be treated as a rough student-level feasibility estimate, not a validated industrial business plan. The biological and financial conclusions depend heavily on assumptions that would need lab-scale data, pilot-scale runs, supplier quotes, GMP planning, and regulatory analysis before the design could be considered realistic.